酵母菌中氧化鯊烯-羊毛硬脂醇環化酵素的突變效應研究

Abstract

在哺乳動物與蕈類中氧化鯊烯環化酵素（OSC）催化2,3-氧化鯊烯成為羊毛硬脂醇的酵素環化反應，和植物中環阿屯醇環化酵素（CAS）負責氧化鯊烯成為環阿屯醇，和細菌中鯊烯-蛇麻烯環化酵素（SHC）負責鯊烯成為蛇麻烯，各為極端複雜的單一步驟轉化反應，其中參與相同或類似的受質並轉變為許多具共通環型結構的產物，顯示這些蛋白質彼此互相擁有精準的分子互動機制，經由發散演化中繼承而來。根據先前突變CAS所表現出產物改變的結果，與SHC結晶結構所推演出的活化中心空腔，顯示在酵母菌的OSC上的Thr384附近可能含有某些會影響反應專一性和陽離子中間物的穩定的重要氨基酸存在，在這份研究中以OSC上從Thr380到Cys392的13個氨基酸殘基為目標區域，利用定點突變分別將每一殘基替代為氨基丙酸，以研究其突變效應。在另一方面，利用隨機突變以尋找其他對酵素活性具有重要影響的可能殘基。 經由將突變株轉殖入去除染色體上erg7基因的酵母菌株，結果在定點突變的Alanine-scanning與隨機突變中均發現當有Trp390Ala突變存在時，變種菌株無法提供基因互補性的環化酵素以支持其存活，進一步考慮Trp390在同源比對時所表現的序列保存性，在TLC分析時未發現任何新產物，以及它在OSC模擬結構上的相對位置，我們可合理地推論Trp390可能以其□-電子在羊毛硬脂醇的碳4位置穩定碳陽子中間物，進而使酵素的活性停止或降低。另外，雖然以E. coli XL1-Red進行的隨機突變所產生的突變無法明白地釐清某些突變株中其他殘基的功能，但其中Trp232與Trp231的功能仍然令人關心而值得在未來進一步探討，因為由初步之結構模擬結果暗示這些殘基可能擔任受質通道的限制作用，且強使氧化鯊烯形成環化反應所必須的不利構形。

The enzymatic cyclization of 2,3-oxidosqualene to lanosterol catalyzed by oxidosqualene-lanosterol cyclase (OSC) in mammals and fungi, oxi-dosqualene to cycloartenol by cycloartenol synthase (CAS) in plant, and squalene to hopene by squalene-hopene cyclase (SHC) in bacteria, constitute extremely impressive one-step conversions that involve same or similar sub-strates transforming into various shared ring-structure products, which indicate precise molecular interaction mechanisms existed mutually in these proteins inherited through divergent evolution. Based on previous results of product alteration exhibited by mutant CAS and active site cavity deduced from crystal structure of SHC, 13 site-directed alanine-scanning mutageneses (sequences 380-392) and a random mutagenesis library from OSC gene of Saccharomyces cerevisiae, respectively, have been generated to study the effect on reaction specificity and intermediates stabilization. All mutant clones were assayed for their ability to complement the cy-clase-deficient, ERG7 knockout, S. cerevisiae strain. Results obtained both from alanine-scanning and random mutageneses clearly showed the failure of complementation of cyclase activity when Trp390 was mutated to alanine, indicating the catalytic function of this residue. Furthermore, results derived from sequence conservation among SHC, CAS and OSC on Trp390, no trace of new product revealed on TLC result, and compatible position between SHC and OSC based on homology modeling, all indicated that the putative role of Trp390 may be involved in stabilizing carbocationic intermediates at atom C4 of lanosterol through its □-electrons. In addition, among mutations arisen from other random mutagenesis, the roles of Trp231 and Trp232 were con-cerned to be examined further that these residues might conduct the constric-tion of substrate passage and compel oxidosqualene into the unfavorable conformation compulsory for cyclization reaction.